Technetium-99m labeled somatostatin-derived peptides for imaging

ABSTRACT

The invention relates to radiolabeled imaging of a mammalian body. The invention in particular provides for reagents labeled with technetium-99m for such imaging. The invention specifically provides somatostatin, somatostatin derivatives, somatostatin analogues or peptides that bind to the somatostatin receptor and contain at least 2 cysteine residues that form a disulfide or wherein the disulfide is reduced to the sulfhydryl form that are directly labeled with technetium-99m and that can be targeted to specific sites within a mammalian body for imaging.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to radiodiagnostic reagents and peptides, andmethods for producing labeled radiodiagnostic agents. Specifically, theinvention relates to technetium-99m (Tc-99m) labeled reagents, methodsand kits for making such reagents, and methods for using such reagents.In particular, the invention relates to Tc-99m labeled somatostatin,derivatives of somatostatin, analogues of somatostatin or peptides thatbind to the somatostatin receptor and contain at least 2 cysteineresidues that form a disulfide or wherein the disulfide is reduced tothe sulfhydryl form.

2. Description of the Prior Art

Somatostatin is a tetradecapeptide that is endogenously produced by thehypothalamus and pancreas in humans and other mammals. The peptide hasthe formula: ##STR1## (Single letter abbreviations for amino acids canbe found in G. Zubay, Biochemistry (2d ed.), 1988, (MacMillanPublishing: New York), p. 33). This peptide exerts a wide variety ofbiological effects in vivo. It is known to act physiologically on thecentral nervous system, the hypothalamus, the pancreas, and thegastrointestinal tract. Somatostatin exerts it effects by binding tospecific receptors expressed at the cell surface of cells comprisingthese organs. In addition, these high-affinity binding sites have beenfound to be abundantly expressed at the cell surface of mostendocrine-active tumors arising from these tissues. Thus, the expressionof high-affinity binding sites for somatostatin is a marker for thesetumor cells, and specific binding with somatostatin can be exploited tolocate and identify tumor cells in vivo.

One method that can readily be adapted to enable detection of tumorcells in vivo based on their expression of high affinity binding sitesfor somatostatin is radioimaging. Radionuclides which emit high energygamma radiation can be readily detected by scintigraphy after injectioninto a human or an animal. A variety of radionuclides are known to beuseful for radioimaging, including ⁶⁷ Ga, ^(99m) Tc (Tc-99m), ¹¹¹ In,¹²³ I, ¹²⁵ I, ¹⁶⁹ Yb or ¹⁸⁶ Re. The sensitivity of imaging methods usingradioactively-labeled peptides is much higher than other techniquesknown in the art, since the specific binding of the radioactive peptideconcentrates the radioactive signal over the cells of interest, forexample, tumor cells. This is particularly important forendocrine-active gastrointestinal tumors, which are usually small,slow-growing and difficult to detect by conventional methods.

Preparation of somatostatin analogues and uses for such analogues isknown in the prior art.

Coy et al., U.S. Pat. No. 4,853,371 disclose synthetic octapeptidesomatostatin analogues.

Coy and Murphy, U.S. Pat. No. 4,871,717 disclose synthetic heptapeptidesomatostatin analogues.

Coy and Murphy, U.S. Pat. No. 4,485,101 disclose synthetic dodecapeptidesomatostatin analogues.

Coy et al., U.S. pat. No. 4,904,642 disclose synthetic octapeptidesomatostatin analogues.

Taylor et al., European Patent Application No. WO 89/04666 disclose amethod of treating cancer in an animal by administering at least aminimal dose of a hexapeptide analog of somatostatin.

Eck and Moreau, European Patent Application No. 90302760.5 disclosetherapeutic octapeptide somatostatin analogues.

Methods for radiolabeling somatostatin analogues that have been modifiedso as to contain a tyrosine amino acid (Tyr or Y) are known in the priorart.

Albert et al., UK Patent Application 8927255.3 disclose radioimagingusing somatostatin derivatives such as octreotide labeled with ¹²³ I.

Bakker et al., J. Nucl. Med. 31: 1501-1509 (1990) describe radioactiveiodination of a somatostatin analog and its usefulness in detectingtumors in vivo.

Bakker et al., J. Nucl. Med. 32: 1184-1189 (1991) teach the usefulnessof radiolabeled somatostatin for radioimaging in vivo.

The use of chelating agents for radiolabeling polypeptides is known inthe prior art.

Byrne et al., U.S. Pat. No. 4,434,151 describe novel homocysteinethiolactone bifunctional chelating agents for chelating radionuclideswhich can couple radionuclides to terminal amino-containing compoundscapable of localizing in an organ or tissue which is desired to beimaged.

Fritzberg, U.S. Pat. No. 4,444,690 describes a series oftechnetium-chelating agents based on 2,3-bis(mercaptoacetamido)propanoate.

Gansow et al., U.S. Pat. No. 4,472,509 teach methods of manufacturingand purifying metal chelate-conjugated monoclonal antibodies.

Byrne et al., U.S. Pat. Nos. 4,571,430 and 4,575,556 describe novelhomocysteine thiolactone bifunctional chelating agents for chelatingradionuclides that can couple radionuclides to terminal amino-containingcompounds capable of localizing in an organ or tissue which is desiredto be imaged.

Nicolotti et al., U.S. Pat. No. 4,861,869 describe bifunctional couplingagents useful in forming conjugates with biologically molecules such asantibodies. This reference describes compounds such asS-benzoylmercaptoacetylglycylglycylglycine.

European Patent Application 84109831.2 describes technetium chelatingcomplexes of bisamido-bisthio-ligands and salts thereof, used primarilyas renal function monitoring agents.

European Patent Application No. 86100360.6 describes dithio, diamino, ordiamidocarboxylic acids or amine complexes useful for making technetiumimaging agents.

European Patent Application 88104755.9 describes various S-protectedmercaptoacetylglycylglycine chelating groups bound to large proteinssuch as antibodies.

Davison et al., Inorg. Chem. 20: 1629-1632 (1981) disclose a novel classof oxotechnetium chelate complexes.

Fritzberg et al., J. Nucl. Med. 23: 592-598 (1982) disclose a technetiumchelating agent based on N,N'-bis(mercaptoacetyl)-2,3-diaminopropanoate.

Byrne and Tolman, J. Nucl. Med. 24: P126 (1983) disclose a bifunctionalthiolactone chelating agent for coupling Tc-99m to biological molecules.

Bryson et al., Inorg. Chem. 27: 2154-2161 (1988) describe thiolateligands for complexing with technetium.

Bryson et al., Inorg. Chem. 29: 2948-2951 (1990) describe thiolateligands for complexing with technetium.

Methods for radiolabeling somatostatin by covalently modifying thepeptide to contain a metal chelating group has been disclosed in theprior art.

Albert et al., UK Patent Application 8927255.3 disclose radioimagingusing somatostatin derivatives such as octreotide labeled with ¹¹¹ Invia a chelating group bound to the amino-terminus.

Albert et al., European Patent Application No. WO 91/01144 discloseradioimaging using radiolabeled peptides related to growth factors,hormones, interferons and cytokines and comprised of a specificrecognition peptide covalently linked to a radionuclide chelating group.

Kwekkeboom et al., J. Nucl. Med. 32: 981 (1991) Abstract #305 relates toradiolabeling somatostatin analogues with ¹¹¹ In.

Albert et al., Abstract LM10, 12th American Peptide Symposium: 1991describe uses for ¹¹¹ In-labeled diethylene-triaminopentaaceticacid-derivatized somatostatin analogues.

Methods for labeling peptides and polypeptides with Tc-99m have beendisclosed in the prior art.

Dean, co-pending U.S. patent application Ser. No. 07/653,012 teachesreagents and methods for preparing peptides comprising a Tc-99mchelating group covalently linked to a specific binding peptide forradioimaging in vivo, and is hereby incorporated by reference.

Fritzberg, U.S. Pat. No. 4,444,690 describes a series oftechnetium-chelating agents based on 2,3-bis(mercaptoacetamido)propanoate.

Gansow et al., U.S. Pat. No. 4,472,509 teach methods of manufacturingand purifying Tc-99m chelate-conjugated monoclonal antibodies.

Reno and Bottino, European Patent Application 87300426.1 discloseradiolabeling antibodies with Tc-99m.

Pak et al., European Patent Application No. WO 88/07382 disclose amethod for labeling antibodies with Tc-99m.

Rhodes, Sem. Nucl. Med. 4: 281-293 (1974) teach the labeling of humanserum albumin with technetium-99m.

Khaw et al., J. Nucl. Med. 23: 1011-1019 (1982) disclose methods forlabeling biologically active macromolecules with Tc-99m.

Byrne and Tolman, supra, disclose a bifunctional thiolactone chelatingagent for coupling Tc-99m to biological molecules.

Cox et al., Abstract, 7th International Symposium on Radiopharmacology,p. 16, 1991, disclose the use of ¹³¹ I- and ¹¹¹ In-labeled somatostatinanalogues in radiolocalization of endocrine tumors in vivo byscintigraphy. Somatostatin labeled with technetium-99m under reducingconditions was used to scintigraphically localize colorectal carcinomain rats following intravenous administration. Tc-99m labeledsomatostatin was prepared by incubating the peptide with a solid phaseelectron donor and sodium pertechnetate at room temperature. Labelingefficiencies of 100% were obtained, with excess free technetium found tobind to the electron donor leaving only labeled complex in solution.Following intravenous administration in rats, rapid blood clearance wasobserved with accumulation in liver, kidneys and bladder; tumor uptakewas found to achieve maximum levels at approximately 4 minpost-injection. Tumor-to-muscle uptake ratios were 5:1 which comparedfavorably with ratios of 3:1 reported for the ¹³¹ I- and ¹¹¹ In-labeledanalogues. The relationship of tumor label uptake to somatostatinbinding was confirmed by a demonstration that somatostatin receptorsblocked with suramin showed suppressed tumor uptake of the label.

The present invention provides peptides which are comprised of between 5and 100 amino acid residues and at least 2 cysteine residues capable offorming a disulfide bond and that are labeled with Tc-99m. The preferredembodiments of the present invention are peptides that are somatostatin,derivatives of somatostatin, analogues of somatostatin or peptides thatbind to the somatostatin receptor and contain at least 2 cysteineresidues that form a disulfide or wherein the disulfide is reduced tothe sulfhydryl form, and that are labeled with Tc-99m. Labeling withTc-99m is an advantage of the present invention because the nuclear andradioactive properties of this isotope make it an ideal scintigraphicimaging agent. This isotope has a single photon energy of 140 keV and aradioactive half-life of about 6 hours, and is readily available from a⁹⁹ Mo-^(99m) Tc generator. Other radionuclides have effective half-liveswhich are much longer (for example, ¹¹¹ In, which has a half-life of60-70 h) or are toxic (for example, ¹²⁵ I). Although Tc-99m is an idealradiolabeling reagent, it has not been widely used in the art prior tothe present invention [see, for example, Lamberts, J. Nucl. Med. 32:1189-1191 (1991)].

Another advantage of the present invention is that theradioactively-labeled somatostatin, somatostatin derivatives,somatostatin analogues or peptides that bind to the somatostatinreceptor and contain at least 2 cysteine residues that form a disulfideor wherein the disulfide is reduced to the sulfhydryl form provided bythe invention have Tc-99m covalently linked to both of thecysteine-derived sulfur atoms capable of forming a disulfide bond of thepeptide. Thus, the label can be directly linked to the peptide. Theadvantage over the prior art is that the invention avoids the necessityof providing somatostatin analogues which contain at least one tyrosineresidue to enable radioactive iodine labeling. The invention also doesnot require the covalent linkage of heterologous chelating groups,described in the prior art. In addition, peptides according to thepresent invention can be prepared according to the methods of theinvention from any available source of somatostatin, somatostatinderivative, somatostatin analog or peptide that binds to thesomatostatin receptor and contains at least 2 cysteine residues thatform a disulfide or wherein the disulfide is reduced to the sulfhydrylform, without the need to synthesize a derivative of a particular designor having specific metal ion chelating properties in addition to itsbiological specificity. The use of native somatostatin enabled by theinvention also provides labeled peptides of well characterized bindingspecificity in vivo.

Thus the present invention provides Tc-99m radiolabeled peptides andradioimaging agents related to somatostatin and its derivatives oranalogues, or indeed any peptide that binds to the somatostatin receptorand contains at least 2 cysteine residues that form a disulfide orwherein the disulfide is reduced to the sulfhydryl form, for use inimaging tumors and other tissues in vivo. The advantages of theinvention include the use of Tc-99m as radionuclide, radiolabeling ofnative as well as modified somatostatin directly via the sulfhydrylgroups of at least 2 cysteine residues of the peptide and the ability toutilize any available source of somatostatin, somatostatin derivative,somatostatin analog or peptide that binds to the somatostatin receptorand contains at least 2 cysteine residues that form a disulfide orwherein the disulfide is reduced to the sulfhydryl form, withoutrequiring any particular modifications of the peptide.

SUMMARY OF THE INVENTION

The invention encompasses somatostatin and peptide derivatives ofsomatostatin labeled with technetium-99m (Tc-99m) useful for imagingtarget sites within a mammalian body. The invention also encompassesmethods for making (Tc-99m)-labeled somatostatin, derivatives ofsomatostatin, analogues of somatostatin or peptides that bind to thesomatostatin receptor and contain at least 2 cysteine residues that forma disulfide or wherein the disulfide is reduced to the sulfhydryl form,and methods for using (Tc-99m)-labeled somatostatin and peptidederivatives of somatostatin to image target sites within a mammalianbody. The invention also includes complexes of Tc-99m with somatostatin,somatostatin derivatives, somatostatin analogues or peptides that bindto the somatostatin receptor and contain at least 2 cysteine residuesthat form a disulfide or wherein the disulfide is reduced to thesulfhydryl form, methods for making such complexes and methods for usingthese complexes to image target sites within a mammalian body.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides technetium-99m labeled peptides forimaging target sites within a mammalian body that comprise between 5 and100 amino acid residues and at least 2 cysteine residues capable offorming a disulfide bond, reacted with technetium-99m under reducingconditions. For purposes of this invention, the phrase "under reducingconditions" is intended to describe reaction of the peptides of theinvention with technetium-99m in the presence of a reducing agent; inpreferred embodiments, the reducing agent is either stannous chloride ora solid-phase reducing agent. Alternatively, the peptides of theinvention are reacted with the reducing agent prior to reaction withtechnetium-99m, so that the peptides are reacted with technetium-99m ina reduced form. In another alternative, both the peptides of theinvention and technetium-99m are reacted with a reducing agent prior tobeing reacted with each other; preferred reducing agents are stannouschloride and a solid-phase reducing agent. The methods of the inventionare also intended to encompass reaction of pre-reduced or unreducedtechnetium-99m in the presence of a reducing agent with peptidescontaining native sulfhydryl groups in the reduced form. All of thesereaction conditions are intended to be described by the phrase "underreducing conditions".

The invention particularly provides radioactively-labeled somatostatin,somatostatin derivatives, somatostatin analogues or peptides that bindto the somatostatin receptor. Somatostatin provided by the inventionincludes commercially available preparations of somatostatin as well assomatostatin prepared as described in Example 1 hereinafter.Somatostatin derivatives, analogues and peptides that bind to thesomatostatin receptor include but are not limited to any peptidesequence comprised of between 5 and 100 amino acid residues and at least2 cysteine residues separated by at least one other amino acid andcapable of forming a disulfide bond, and exhibiting the biologicalbinding properties of native somatostatin or any modification of thoseproperties, wherein the peptide retains specific binding characteristicsthat are identical to, similar with or distinct from the nativesomatostatin peptide. Derivatives are intended to include modificationsin the composition, identity and derivitization of the amino acidcomponents of the peptide, provided that the peptide sequence iscomprised of at least 2 cysteine residues separated by at least oneother amino acid and capable of forming a disulfide bond, and thepeptide retains the specific biological binding properties to thesomatostatin receptor of native somatostatin. These modificationsinclude compositions comprised of the D- as well as the nativeL-stereoisomers of any of the component amino acids of the peptide;substitution in the aromatic sidechain of any component amino acid (forexample, F, Y or W); derivitization of the amino- or carboxyl-groups inthe sidechains of any component amino acid (for example, D, E, K, N orQ), or substitutions in the amino-or carboxyl-terminus of the peptide,or wherein the peptide is covalently linked at either the amino- orcarboxy-terminus to any other protein, peptide or biologically activemolecule, or cyclization of the peptide. (Single letter abbreviationsfor amino acids can be found in G. Zubay, supra). Somatostatinderivatives are intended to include peptides comprised of amino acidswhich are naturally occurring or not naturally-occurring amino acids.Although the present invention is specifically intended to relate toradiolabeling of somatostatin, somatostatin derivatives, somatostatinanalogues or peptides that bind to the somatostatin receptor and containat least 2 cysteine residues capable of forming a disulfide bond, themethods of the invention are applicable to any peptide or polypeptideprovided the peptide or polypeptide is comprised of at least 2 cysteineresidues separated by at least one other amino acid and capable offorming a disulfide bond, and the scope of the invention is intended toencompass such other peptides and polypeptides.

The invention provides Tc-99m labeled somatostatin, somatostatinderivatives, somatostatin analogues or peptides that bind to thesomatostatin receptor that are labeled as a result of the formation ofcoordinate covalent linkage of the Tc-99m atom to 2 of thecysteine-derived sulfur atoms comprising the cysteine residues of thepeptide. This method of directly labeling somatostatin or somatostatinderivatives, analogues or related peptides is advantageous over themethods known in the prior art. Methods of radioactively labelingsomatostatin or related peptides known prior to the present inventionrequired the use of substituted somatostatin derivatives that werecomprised of at least one tyrosine residue in order to permitradioiodination of the peptide. Alternatively, radiolabeled somatostatinpeptides have been made by covalent linkage of a metal chelating groupto the amino terminus of the peptide and reaction with the radionuclide.A particular disadvantage of the covalent attachment of these chelatinggroups is that the presence of these groups at the amino terminus of thepeptide might interfere with the biological properties of the peptidesin vivo and in addition may intrinsically cause biocompatabilityproblems either itself or when linked to another peptide. Labeledpeptides of the present invention can be prepared directly fromcommercially available somatostatin or any somatostatin derivative,analog or related peptide of sufficient purity or synthesized by meanswell known to those with skill in the art. An additional advantage ofthe peptides of this invention and those prepared by the methods of theinvention is that these peptides contain Tc-99m covalently linked to thepeptides, thus providing a reagent with maximum stability due to thenature of the Tc-peptide bond.

In forming a complex of radioactive technetium with the peptides of thisinvention, the technetium complex, preferably a salt of Tc-99mpertechnetate, is reacted with the peptides of this invention in thepresence of a reducing agent; in a preferred embodiment, the reducingagent is stannous chloride. In an additional preferred embodiment, thereducing agent is a solid-phase reducing agent. Complexes and means forpreparing such complexes are conveniently provided in a kit formcomprising a sealed vial containing a predetermined quantity of thesomatostatin, somatostatin derivatives, somatostatin analogues orpeptides that bind to the somatostatin receptor and contain at least 2cysteine residues that form a disulfide or wherein the disulfide isreduced to the sulfhydryl form that are to be labeled and a sufficientamount of reducing agent to label the peptide with Tc-99m.Alternatively, the complex may be formed by reacting the peptides ofthis invention with a pre-formed labile complex of technetium andanother compound known as a transfer ligand. This process is known asligand exchange and is well known to those skilled in the art. Thelabile complex may be formed using such transfer ligands as tartrate,citrate, gluconate or mannitol, for example. Among the Tc-99mpertechnetate salts useful with the present invention are included thealkali metal salts such as the sodium salt, or ammonium salts or loweralkyl ammonium salts. The reaction of the peptides of this inventionwith Tc-pertechnetate or preformed Tc-99m labile complex can be carriedout in an aqueous medium at room temperature. The anionic complex whichhas a charge of [-1] is formed in the aqueous medium in the form of asalt with a suitable cation such as sodium cation, ammonium cation,mono, di- or tri-lower alkyl amine cation, etc. Any conventional salt ofthe anionic complex with a pharmaceutically acceptable cation can beused in accordance with this invention.

In another embodiment of the present invention, the somatostatin,somatostatin derivatives, somatostatin analogues or peptides that bindto the somatostatin receptor and contain at least 2 cysteine residuescapable of forming a disulfide bond that are to be labeled are reducedprior to labeling by incubating the peptides with a reducing agent. In apreferred embodiment, the reducing agent is stannous chloride. In anadditional preferred embodiment, the reducing agent is a solid-phasereducing agent. The pre-reduced peptide is then labeled by reaction witha Tc-99m under reducing conditions or with pre-reduced Tc-99m or Tc-99mcomplex.

In a preferred embodiment of the invention, a kit for preparingtechnetium-labeled peptides is provided. Peptides that are somatostatin,somatostatin derivatives, somatostatin analogues or peptides that bindto the somatostatin receptor and contain at least 2 cysteine residuescapable of forming a disulfide bond are chemically synthesized usingmethods and means well-known to those with skill in the art anddescribed hereinbelow in Example 1. Peptides thus prepared are comprisedof at least 2 cysteine residues, wherein the sulfhudryl groups of thecysteine residues are in the reduced form. An appropriate amount of thepeptide is introduced into a vial containing a reducing agent, such asstannous chloride or a solid-phase reducing agent, in an amountsufficient to label the peptide with Tc-99m. An appropriate amount of atransfer ligand as described (such as tartrate, citrate, gluconate ormannitol, for example) can also be included. Technetium-labeled peptidesaccording to the present invention can be prepared by the addition of anappropriate amount of Tc-99m or Tc-99m complex into the vials andreaction under conditions described in Example 2 hereinbelow.

Radioactively labeled peptides provided by the present invention areprovided having a suitable amount of radioactivity. In forming theTc-99m radioactive anionic complexes, it is generally preferred to formradioactive complexes in solutions containing radioactivity atconcentrations of from about 0.01 millicurie (mCi) to 100 mCi per ml.

Technetium labeled somatostatin, somatostatin derivatives, somatostatinanalogues or peptides that bind to the somatostatin receptor can be usedfor visualizing organs such as the kidney for diagnosing disorders inthese organs, and tumors, in particular gastrointestinal tumors,myelomas, small cell lung carcinoma and other APUDomas, endocrine tumorssuch as medullary thyroid carcinomas and pituitary tumors, brain tumorssuch as meningiomas and astrocytomas, and tumors of the prostate,breast, colon, and ovaries can also be imaged. In accordance with thisinvention, the technetium labeled peptides or anionic complexes eitheras a complex or as a salt with a pharmaceutically acceptable cation areadministered in a single unit injectable dose. Any of the commoncarriers such as sterile saline solution, plasma, etc., can be utilizedafter the radiolabeling for preparing the injectable solution todiagnostically image various organs, tumors and the like in accordancewith this invention. Generally, the unit dose to be administered has aradioactivity of about 0.01 mCi to about 100 mCi, preferably 1 mCi to 20mCi. The solution to be injected at unit dosage is from about 0.01 ml toabout 10 ml. After intravenous administration, imaging of the organ ortumor in vivo can take place in a matter of a few minutes. However,imaging can take place, if desired, in hours or even longer, afterinjecting into patients. In most instances, a sufficient amount of theadministered dose will accumulate in the area to be imaged within about0.1 of an hour to permit the taking of scintiphotos. Any conventionalmethod of imaging for diagnostic purposes can be utilized in accordancewith this invention.

The technetium labeled peptides and complexes may be administeredintravenously in any conventional medium for intravenous injection suchas an aqueous saline medium, or in blood plasma medium. Such medium mayalso contain conventional pharmaceutical adjunct materials such as, forexample, pharmaceutically acceptable salts to adjust the osmoticpressure, buffers, preservatives and the like. Among the preferred mediaare normal saline and plasma.

The methods for making and labeling these peptides are more fullyillustrated in the following examples. These examples are provided forillustrating the invention and are not meant to be construed aslimiting.

EXAMPLE 1 Solid Phase Peptide Synthesis

Solid phase peptide synthesis (SPPS) was carried out on a 0.25 millimole(mmole) scale using an Applied Biosystems Model 431A Peptide Synthesizerand using 9-fluorenylmethyloxycarbonyl (Fmoc) amino-terminus protection,coupling with either dicyclohexylcarbodiimide/hydroxybenztriazole(DDC/HOBT) or 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate/hydroxybenztriazole (HBTU/HOBT), andp-hydroxymethylphenoxymethylpolystyrene (HMP) resin forcarboxyl-terminus acids or Rink amide resins for carboxyl-terminusamides. Resin-bound products were routinely cleaved using a solution ofcomprised of trifluoroacetic acid, water, phenol, thioanisole, andethanedithiol, prepared in ratios of 90:5:7.5:5:2.5 for 1.5-3 h at roomtemperature. Crude peptides were purified by preparative high pressureliquid chromatography (HPLC) using a Waters Delta Pak C18 column andgradient elution using 0.1% trifluoroacetic acid (TFA) in water modifiedwith acetonitrile. Acetonitrile was evaporated from the eluted fractionswhich were then lyophilized. The identity of each product was confirmedby fast atom bombardment mass spectroscopy (FABMS).

Acyclopeptides thus prepared may be then cyclized by the formation of anintramolecular disulfide bond either before or after HPLC purificationin solution at pH 8.0 either chemically using K₃ Fe(CN)₆ orspontaneously catalyzed by air. Cyclized peptides are purified by HPLCas described above.

EXAMPLE 2 Somatostatin Labeling with Technetium-99m

Somatostatin was labeled with technetium-99m (Tc-99m) using thefollowing protocols.

Direct Reduction Method. 0.66 μmoles somatostatin-14 (S-14=Formula I;Bachem Bioscience Inc., Cat# H-1490) were reduced using an immobilizedreductant (Reduce-Imm™ Reducing Kit, Pierce Chemical Co., Cat# 77700H),a reagent for column-based reduction of disulfides consisting of aproprietary solid phase reductant cross-linked to 6% beaded agarose.Disulfide reduction using this reagent results in a 98.6% yield ofexpected reduced thiol (SH) as determined by Ellman's assay [Ellman,Arch. Biochem. Biophys. 74: 443 (1958)]. (Tc-99m)-labeling wasaccomplished by incubating 100 nM SH equivalents of S-14 (117 μl) with0.6 mCi (29 μl) Tc-99m gluceptate at room temperature for 15 min. Tc-99mgluceptate was prepared by reconstituting a Glucoscan Vial (E.I. DuPontDeNemours, Inc. Wilmington, Del.) with 1.0 ml Tc-99m sodiumpertechnetate containing 21 mCi.

The extent of Tc-99m peptide labeling achieved was determined by thinlayer chromatography (TLC) using Merck silica gel 60 F₂₅₀aluminum-backed strips spotted with 10 μl of sample and chromatographedwith acetone or phosphate buffered saline (PBS). Under these conditions,99% of (Tc-99m)-associated radioactivity remained at the origin (R_(f)=0.0) in either solvent, indicating that no significant concentration offree Tc-99m pertechnetate or Tc-99m gluceptate could be detected in thesample. Purity of the 99m-Tc-labeled peptide was determined by HPLCusing a Vydak 218TP54 analytical column (RP-18, 5 micron, 250 mm long)eluted with a gradient that ranged from 0% Solution A (CH₃ CN:H₂ O:TFA,90:10:0.1) and 100% Solution B (0.1% TFA in water) to 100% Solution Aand 0% Solution B achieved over 10 min, with conditions of 100% SolutionA and 0% Solution B maintained for the remainder of chromatographic run.An in-line NaI detector was used to determine that 98.9% of theradiometric species existed as a single peak with a retention time of14.5 min. In contrast, unconjugated Tc-99m pertechnetate and Tc-99mgluceptate eluted from this column within 1-4 min under theseconditions, confirming the successful labeling of somatostatin withTc-99m using this method.

Stannous Chloride Method. A stannous chloride solution was freshlyprepared by dissolving 26.5 mg SnCl₂ in 1.128 ml 1N HCl. A gluconatesolution was prepared by dissolving 500 mg of the potassium salt ofD-gluconic acid in 50 mM phosphate buffer and adjusting the pH to 7.4with 1N NaOH or 1N HCl. The gluconate solution was deoxygenated bybubbling treatment with nitrogen gas. 17 μl (equivalent to 400 μg) ofthe stannous chloride solution plus 17 μl 1N NaOH were added to 1.0 mlof the gluconate solution to prepare the peptide reconstitutionsolution.

55.5 nmoles of S-14 (0.09 mg) were dissolved in 1.0 ml of the peptidereconstitution solution prepared as described above and then incubatedfor 3 h at room temperature prior to the addition of 250 μl Tc-99m (6.5mCi) sodium pertechnetate (sample #1).

Alternatively, 86.3 nmoles of S-14 (0.14 mg) were dissolved in 1.0 ml ofthe peptide reconstitution solution 5 minutes prior to the addition of250 μl Tc-99m (6.5 mCi) sodium pertechnetate (sample #2).

Somatostatin in both samples was labeled with Tc-99m by incubating thesamples at room temperature for 60 min. The extent of Tc-99m peptidelabeling was determined by TLC using Merck silica gel 60 F₂₅₀aluminum-backed strips spotted with 10 μl of sample and chromatographedin acetone or PBS. After 60 min, both samples exhibited at least 94% ofthe radioactivity at the origin (R_(f) =0.0) in both solvents,demonstrating that no significant concentration of free Tc-99mpertechnetate or Tc-99m gluconate could be detected in either sample.

Tc-99m purity was determined by HPLC using a Vydak 218TP54 analyticalcolumn and eluted as described above. An in-line NaI detector was usedto evaluate the distribution of radiometric species in each sample.Sample #1 exhibited 91% of detected radioactivity as a single specieswith a retention time equivalent to 14.5 min. Sample #2 exhibited 96% ofdetected radioactivity as 4 separate species with retention times of19.4 min (68% of total detected radioactivity), 20.2 min (17%), 20.9 min(7%), and 21.5 min (4%). Unconjugated Tc-99m pertechnetate and Tc-99mgluconate elute within 1-4 minutes under these conditions. These resultsconfirm the successful labeling of somatostatin with Tc-99m using eithervariation of this method.

What is claimed is:
 1. A reagent for preparing a technetium-99m labeledpeptide for imaging target sites within a mammalian body, the reagentcomprising between 5 and 100 amino acid residues and at least 2 cysteineresidues capable of forming a disulfide bond, wherein the reagent isdirectly labeled with technetium-99m by reaction with technetium-99munder reducing conditions.
 2. The reagent of claim 1 that issomatostatin, a derivative of somatostatin, an analog of somatostatin ora peptide that binds to the somatostatin receptor.
 3. A complex formedby reacting somatostatin, a derivative of somatostatin, an analog ofsomatostatin or a peptide that binds to the somatostatin receptor andcontains at least 2 cysteine residues that form a disulfide or whereinthe disulfide is reduced to the sulfhydryl form, with technetium-99m inthe presence of a reducing agent.
 4. The complex of claim 3, wherein thereducing agent is a stannous ion or a solid-phase reducing agent.
 5. Acomplex formed by ligand exchange of a prereduced technetium-99m complexwith somatostatin, a derivative of somatostatin, an analog ofsomatostatin or a peptide that binds to the somatostatin receptor andcontains at least 2 cysteine residues that form a disulfide or whereinthe disulfide is reduced to the sulfhydryl form.
 6. A method for imaginga target site within a mammalian body comprising:(a) administering aneffective diagnostic amount of a peptide that is somatostatin, aderivative of somatostatin, an analog of somatostatin or a peptide thatbinds to the somatostatin receptor and contains at least 2 cysteineresidues that form a disulfide or wherein the disulfide is reduced tothe sulfhydryl form which is directly labeled with technetium-99m,wherein the labeled peptide binds to the target site; and (b) detectingthe localized technetium-99m.